Purification and characterization of a maturation-activated myelin basic protein kinase from sea star oocytes.
A meiosis-activated myelin basic protein (MBP) kinase was purified approximately 8700-fold from soluble post-germinal vesicle breakdown extracts from maturing oocytes of the sea star Pisaster ochraceus. Purification to apparent homogeneity was achieved by sequential chromatography on DEAE-cellulose, hydroxylapatite, phosphocellulose, phenyl-Sepharose, heparin-Sepharose, polylysine-Sepharose, and Mono-Q. The final product exhibited an apparent molecular mass of approximately 42 kDa by both native gradient and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and this precisely correlated with the chromatographic behavior of the recovered MBP kinase activity on a Superose 6/12 column. The kinase utilized the MBP as the major substrate with little or no phosphorylation of histones (H1, H2A, or H2B), casein, phosvitin, protamine, or 40 S ribosomal proteins. The purified enzyme was relatively insensitive to high concentrations of beta-glycerol phosphate, calmodulin, EGTA, NaCl, sodium fluoride, dithiothreitol, spermine, and heparin but was quite sensitive to inhibition by metal ions such as Mn2+, Zn2+, and Ca2+. The true Km values for ATP and myelin basic protein were determined to be 58 and 25 microM, respectively, using double-reciprocal plots. The purified enzyme was unable to utilize GTP in place of ATP. The enzyme was shown to rapidly undergo autophosphorylation. The autophosphorylation was sensitive to alkali treatment implying that phosphate was incorporated on serine/threonine residues. The properties of this MBP kinase are reminiscent of a protein kinase that is also activated in a cyclic fashion at M-phase during the early cell divisions of sea star and sea urchin embryos (Pelech, S. L., Tombe, R., Meijer, L., and Krebs, E. G. (1988) Dev. Biol. 130, 26-36).
Sanghera
J S
JS
Biomedical Research Center, University of British Columbia, Vancouver, Canada.
Paddon
H B
HB
Bader
S A
SA
Pelech
S L
SL
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent; D002845:Chromatography; D004789:Enzyme Activation; D005260:Female; D038362:Glycogen Synthase Kinase 3; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D008540:Meiosis; D008970:Molecular Weight; D009865:Oocytes / Q000201:enzymology* / Q000502:physiology; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D013215:Starfish; D013379:Substrate Specificity; D015032:Zinc / Q000494:pharmacology
D002413:Cations, Divalent; D047428:Protein Kinase Inhibitors; D008345:Manganese; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D038362:Glycogen Synthase Kinase 3; D015032:Zinc; D002118:Calcium